The present invention relates to new immortalised cell lines derived from normal human skin tissues and presenting improved differentiation characteristics, to a new method for producing these cell lines and to various usages thereof, particularly in the field of creating artificial skin.
The production of immortalised cell lines derived from normal human skin tissues has already been described. In general, the processes used for this purpose comprise transformation of human skin cells, for example of keratinocytes and melanocytes, which are cultivated in vitro with agents conveying immortalization. Immortalization relates to the production of cells, which may be cultivated during prolonged period of times in vitro, theoretically for an indefinite period. These cells are also designated continuous cell lines. In contrast thereto, the non-immortalised cells are only capable to proliferate during a defined number of cell divisions in vitro. The immortalised cells are extremely advantageous, since they provide a stable, potentially unlimited source of cells having defined characteristics. Typical agents for the production of immortalised cell lines and of immortalised human skin cell lines comprising in particular, for example viruses, recombinant viruses and plasmids containing DNA sequences conveying the property of immortalization.
The most common process for producing immortalised human cell lines comprises probably the use of sequences of the Simian Virus 40 (SV 40) and more specifically of the DNA of the large T antigen (T-Ag) of SV40 as the agent of immortalization. For example, Steinberg et al., J. Cell Phys, 123, 117-125 (1985); U.S. Pat. No. 4,885,238 (Reddel et al.); U.S. Pat. No. 4,707,448 (Major); Stoner et al., Cancer Res., 51, 365-371 (1991); Chopra et al., In vitro Cell Dev. Biol., 30A, 539-546 (1994); Chopra et al., In vitro Cell Dev. Biol., 27A, 763-765 (1991); Christian et al., Cancer Res., 47, 6066-6073 (1987); Rhim et al., Science, 227, 1250-1252 (1985); and Grubman et al., Gastrointest. Liver Physiol., 29, G1060-G1070 (1994) report of the use of SV40 vectors and of vectors containing the sequence of the large T antigen of SV40 for producing immortalised human cell lines. The introduction of such sequences is generally effected by infection with the SV40 virus or with a 12/SV40 hybrid-adenovirus or by transfection of cells with a recombinant plasmid containing the long terminal repeats of the Rous sarcoma virus and the regulatory SV40 ori-region by coprecepitation in the presence of strontium phosphate (see Brash et al., Mol. Cell Biol.,7, 2031-2034, 1987).
Another known process for the production of immortalised cell lines and in particular of immortalised human keratinocytes comprises the transfection or infection of cells with DNA sequences of the human papilloma virus (HPV). For example, U.S. Pat. No. 5,376,542 (Schlegel) describes the immortalization of human epithelial cells with the E6 and E7 genes isolated of HPV-16, 18, 31, 33 or 35 or with the E7 gene alone for producing non-tumorigenic immortalised human cell lines. Moreover, Barbosa et al. Oncogene, 4, 1529-1532 (1989); and Mxc3xcnger et al. J. Virol., 63(10): 4417-4421 (1989) report of the use of the E6 and E7 genes of HPV-16 and HPV-18 for producing immortalised human keratinocytes. Moreover, Dxc3xcrst et al., Oncogene, 1, 251-256 (1987) describes the immortalization of keratinocytes with the papilloma virus type 16.
Nevertheless, although numerous groups described immortalized keratinocyte cell lines and their use in in vitro assays, the immortalized keratinocyte cell lines of the state of the art present usually one or more properties rendering their utilization disadvantageous. For example, the previously described immortalized keratinocytes present one or more of the following properties: (i) reduction or loss of expression of differentiation markers, for example of proteins being expressed by normal differentiated keratinocytes, (ii) properties of modified growth in culture of tissues and (iii) formation of a stratified and polarized epithelium having a stratum corneum para-keratinocyte.
In order to eliminate these disadvantages, EP780469 (Socixc3xa9txc3xa9 des Produits Nestlxc3xa9) proposes a new method for immortalizing keratinocytes or human melanocytes using en sus a new culture medium, the vector pLXSHD+SV40(#328), which is derived from SV40 virus, or equally the vector pLXSHD+E6/E7, which is derived from the human papilloma virus 16 (HPV-16). The immortalized cells obtained in this way preserve the capacity of differentiation and of expression of proteins and of enzymes, which are expressed by normal differentiated keratinocytes and melanocytes, even after an elevated number of passages in culture.
However, in spite of the previous descriptions, there is still an important need in the practical field to have human immortalised keratinocytes possessing even more ameliorated properties. Such cells would be extremely advantageous for numerous uses, particularly for analyses needing highly differentiated skin cells.
For this purpose, the present invention relates to a human immortalized keratinocyte cell line by means of at least one tumorgenic functional gene of DNA viral origin, which is characterized in that it:
(1) is non-tumorigenic,
(2) conserves the capacity to differentiate and to express proteins and enzymes expressed by normal differentiated keratinocytes after an elevated number of passages in tissue culture, and
(3) forms a stratified and polarised epithelium having a stratum corneum ortho-keratinocyte, if cultivated in organo-typical culture in a serum-free medium and without a layer of nourishing cells.
Another object of the present invention is to provide a new process for producing immortalised keratinocyte cell lines derived from normal skin tissues.
Yet, another object of the present invention is to provide processes for the use of these keratinocyte cell lines according to the invention, for example for immunological, pharmacological, photo- and chemical-toxological analyses of skin reaction, and for the expression of heterologous genes.